FIGURE 5.

HPLC analysis of the synthetic substrate PL405 cleavage by ECE-2. The HPLC conditions were Kromasil C₁₈ column (5 μm, 100 Å, 4.6 × 150 mm); gradient 10–90% solvent B in 60 min. Solvent A was 0.1% trifluoroacetic acid in H₂O. Solvent B was 0.1% trifluoroacetic acid in CH₃CN. The flow rate was 1 ml/min. Absorbance detection (UV) was 343 nm. A, injection of 100 μl of 10 μm of PL405 substrate in 200 mm sodium acetate, pH 5.5, 0.2 n HCl. The peak of the substrate has a retention time of 27.8 min. B, injection of 100 μl of 10 μm of Ac-SKG-Pya cleavage product PL415 in the same buffer. The retention time of the peak was 23.1 min. C, the PL405 substrate (20 μm) was incubated 3 h at 37 °C in 200 mm sodium acetate pH 5.5 with 500 ng/ml of ECE-2. The reaction was stopped with 0.2 n HCl, and 100 μl of the reaction were separated by HPLC.